Chromatography is the most effective means among methods for the analysis of the components of a mixture and their contents and for their separation and purification. Chromatography performs the separation of different substances by utilizing the substance-specific distribution ratio (also understood as the adsorption equilibrium) between a porous solid (the stationary phase) that is spatially immobilized in a column or a tube known as a capillary, and a fluid (the moving phase) that moves in the spaces in the porous solid. Gas chromatography and liquid chromatography are typical here. A gas is used as the moving phase in the former.
However, in order for a separation target to move mixed in a gas phase, at least a certain vapor pressure must be exhibited, and as a consequence only a relatively limited range of analytes, i.e., that have a low molecular weight and that lack charge, can be employed. Liquid chromatography, on the other hand, uses a liquid as the moving phase and can be applied to most substances assuming the selection of a suitable moving phase. Still, since liquids generally have high viscosities, limits are imposed by the increase in the viscous resistance when the generation of excellent separation is sought using a long column or capillary.
Supercritical fluid chromatography (SFC) was invented as a technology that can overcome the shortcomings of both gas chromatography and liquid chromatography. Supercritical fluid chromatography utilizes the characteristics of a supercritical or subcritical fluid, i.e., it dissolves other compounds much better than a gas and has a lower viscosity and a higher diffusion rate than a liquid. SFC using carbon dioxide as the supercritical fluid is generally employed based on safety and device considerations, and its use is gradually becoming more widespread. In addition to SFC, chromatography that uses electrical attraction and so-called thin-layer chromatography (a variant of liquid chromatography), in which paper or particles are consolidated in a thin layer, are available, but their range of application is not very broad.
The typical modes for liquid chromatography are normal-phase chromatography, which uses the combination of a high-polarity stationary phase and a low-polarity stationary phase, and reversed-phase chromatography, in which these polarities are reversed. HILIC, in which both phases are polar, has also been receiving attention quite recently. In addition, chromatographies based on specific interactions are also known, such as ligand-exchange chromatography, which utilizes metal ion/ligand interactions, and affinity chromatography, which utilizes biochemical interactions. Their characteristics and separation mechanisms are generally understood, and their technical advances mainly concern improvements in particle shape in order to improve the separation efficiency.
In contrast, the characteristics of supercritical fluid chromatography (SFC) are reported to be similar to those of normal-phase chromatography. However, many aspects of its characteristics and mechanisms are still not well understood.
The stationary phases used in conventional liquid chromatography (HPLC) have generally been also utilized as the stationary phase (also referred to as the column packing) in SFC. For example, as introduced in Nonpatent Document 1, these are silica gels or silica gels that have undergone surface modification with various atomic groups.
The modifying group may contain a saturated alkyl chain in various chain lengths; or may be a modifying group in which a condensed polycyclic aromatic hydrocarbon group or one or two benzene rings are bonded via an alkyl chain or an alkyl chain that includes the amide bond or ether linkage; or a modifying group in which the characteristic feature is a halogen-substituted benzene ring; or a modifying group in which a halogenated alkyl group is bonded; or a modifying group in which a polar group, e.g., the 2,3-dihydroxypropyl group, CN group, or NH2 group is bonded; or may be a high molecular weight modifying group in the form of crosslinked polystyrene, polyvinyl alcohol, or polyethylene glycol. In addition, carbon having a graphite structure is also a special stationary phase. Among these, (2-pyridyl)ethyl group-bonded stationary phases, referred to as 2-ethylpyridine, in particular are frequently used in SFC; their use is preferred because they provide sharp peak elution even for basic compounds, which undergo tailing and give broad peaks with ordinary stationary phases.
However, as nonetheless indicated in Nonpatent Document 2, the retention trends for various compounds are similar and not a few stationary phases also exhibit no difference in characteristics. It is within this context that the present inventors, recognizing that the ability to discriminate among molecules having similar structures is a necessary condition, have diligently pursued the development of SFC stationary phases.
On the other hand, polysaccharide-type stationary phases for chiral separation are also used in SFC and are utilized in chiral separations in practice (for example, Nonpatent Document 3). Polysaccharide derivatives are also provided with an excellent capacity to distinguish molecular structures outside of chiral separations, but can be difficult to use, because their selectivity range is too large and the separation of optical isomers becomes entangled.
The present inventors have carried out focused investigations thinking that polymers might have a still-to-be-elucidated specific capacity to discriminate molecules. In relation to polymers provided with such a structure, for example, polyesters, there have been attempts to carry out HPLC using fiber-filled columns, and fibers such as PP, PET, nylon-6, Kevlar (trademark) (polyamide), and cellulose have been disclosed as adsorbents (Nonpatent Document 4 and the references cited therein). However, while these are garnering interest, as seen on page 25 of Nonpatent Document 5, they provide broad peaks in actual chromatography and are not practically usable. In addition, the use of a so-called vinyl polymer, e.g., a divinylbenzene/styrene copolymer and so forth, as packing is disclosed in Patent Document 1.
Patent Document 2 discloses polystyrene beads for polynucleotide separation by liquid chromatography and also provides polyesters as an example thereof. However, when a nonporous spherical body, such as that disclosed in Patent Document 2, is used as a chromatographic stationary phase, retention may occur with relatively strongly polar polymers, such as the polynucleotides that are the separation target in the cited invention, while ordinary low molecular weight compounds cannot be retained—or strong tailing is produced even when retention does occur—and a practical analytical method is thus not obtained. This, which may also be said of the fibrous polymer cited above, is due in the case of a thick nonporous polymer to the time required for the separation target to achieve adsorption equilibrium between the solid phase (the polymer used as the adsorbent) and the liquid phase (the moving phase) since the retention of a low molecular weight compound by a polymer occurs when such a molecule diffuses into the interior of the polymer and the diffusion of molecules within a polymer is generally slow.    Patent Document 1 Japanese Patent No. 3,858,509    Patent Document 2 Japanese Translation of PCT Application No. 2002-506426    Non-patent Document 1 C. West et al., J. Chromatogr. A, 1203(2008) 105    Non-patent Document 2 C. West et al., J. Chromatogr. A, 1191(2008) 21    Non-patent Document 3 Y. Kaida et al., Bull. Chem. Soc. Jpn., 65, 2286(1992)    Non-patent Document 4 R. K. Marcus, J. Separation Science, 31, 1923(2008)    Non-patent Document 5 R. K. Marcus et al., J. Chromatogr. A, 986, 17(2003)